Amino acids are the building blocks of proteins, and certain amino acids participate in critical biochemical and cellular processes essential for the growth, development and survival of an organism. Qualitative and quantitative amino acid analysis is, therefore, an important analytical and diagnostic tool in a wide variety of clinical, biopharmaceutical and agriculture applications as well as in metabolic and metabolomic research studies.
The presence, absence, identity, amount or modification of an endogenous amino acid as well as its presence and amount in comparison to other amino acids (i.e., the overall profile of free amino acids) are important parameters in assessing a subject's metabolic state. Aberrant amino acid levels or increased/decreased levels of certain amino acids in comparison to other amino acids can indicate a metabolic disturbance requiring precise and accurate detection and quantitation so that an appropriate intervention can be devised. Therapeutic interventions of metabolic disturbances often include dietary restriction, but can also involve the administration of vitamins and/or other pharmacological agents. Depending on the condition, these treatments may be essential for a patient's survival and optimal mental development.
The qualitative and quantitative analysis of free amino acids in biological fluids and tissues is central to the diagnosis and management of a wide variety of metabolic disturbances including primary amino acid enzymopathies (e.g., phenylketonuria, maple syrup urine disease) and disorders of amino acid transport (e.g., cystinuria). Comprehensive metabolic profiling gives a snapshot of the current physiological state of a subject or experimental organism and, besides uncovering primary metabolic disturbances, is instrumental in evaluating a subject's nutritional status, organ function and compliance with metabolic therapies.
A number of semi-automated High Pressure Liquid Chromatography (HPLC) systems for amino acid analysis have been developed for use in the clinical setting, using pre-column and/or post-column derivatization techniques. The traditional and most widely used approach is based on separation by cation-exchange HPLC and post-column derivatization with ninhydrin, a method whereby negatively charged interferents (e.g., chloride, phosphate) are eluted with the void volume (Spackman et al, 1958). Despite overall excellent performance with simple sample preparation, good linearity over a wide dynamic range, and baseline separation of compounds, this method suffers from long sample analysis times as well as the use of costly reagents and buffers. Alternatively, pre-column derivatization with various reagents such as o-phthalaldehyde (OPA) and separation by reversed-phase HPLC achieve high sensitivity and fast analysis times, but require extensive sample preparation. Furthermore, this approach is generally relegated for the analysis of matrices that have few interferents, such as urine. Since the mode of detection of all these methods is based on spectrophotometry, their performance particularly with respect to specificity is generally compromised by potential interferences from co-eluting molecules that cannot adequately be identified or detected by spectrophotometric detection alone.
More recently, methods utilizing tandem mass spectrometry (MS/MS) for amino acid analysis in physiological samples have been reported (Freeto et al, 2007, Dietzen et al, 2008, Casetta et al, 2000, Piraud et al, 2003, Piraud et al, 2005a, Piraud et al, 2005b). These share features with the traditional pre-column and post-column methods, and include analysis of both derivatized and underivatized molecules. While all methods represent significant improvements in specificity and analysis times, those involving separation of derivatized (i.e., butylated) molecules by reversed-phase HPLC are associated with lengthy sample preparation and decreased reproducibility stemming from the derivatization procedure itself. In addition, preparative and chromatographic conditions do not favor the removal of negatively charged molecules (e.g., chloride, phosphate), which lead to ion suppression and consequently diminished sensitivity for some molecules.
Reversed-phase HPLC separation of underivatized amino acids in the presence of the ion-pairing agent tridecafluoroheptanoic acid effectively removes interfering molecules and reduces ion suppression, but the associated solvent conditions still result in reduced sensitivity for the most nonpolar molecules, notably glycine, taurine and s-sulfocysteine, due to poor ionization in the detector (Piraud et al, 2005b). In some cases problems with imprecision are overcome by the inclusion of one or more stable-isotope internal standards, but these reagents are costly and not commercially available for all amino acids.
Improved methods for the sensitive and specific, yet quick and cost-effective analysis of amino acids, in particular of the clinically relevant amino acids, in biological fluids and tissues would greatly facilitate the reliable and rapid detection of metabolic disturbances, assessment of organ function and nutritional status of a subject and are, therefore, urgently needed. Those improved methods would also be valuable for evaluating the amino acid content in nonbiological samples to assess parameters such as quality and purity of a sample.